Protein Analysis

Protein Analysis

• Protein structure can be analyzed using:
  • X-ray crystallography
  • Nuclear magnetic resonance (NMR)
  • Spectroscopy
• Protein activity and amount can be analyzed using:
  • UV spectroscopy
  • Bradford protein assay
  • Coomassie brilliant blue dye

Determination of Protein Primary Structure (Amino Acid Composition)

• Identification of proteins associated with specific physiologic states or diseases is an important goal of molecular medicine
• Primary sequence of a protein provides a molecular fingerprint for identification and information for cloning the gene or genes that encode it

Protein Sequencing

• N-Terminal analysis determines the amino terminal end of the polypeptide chain using:
  • Sanger's method
  • Edman's method
• C-Terminal analysis determines the carboxy terminal end of the polypeptide chain using:
  • Carboxypeptidase method
• Edman's sequencing method involves:
  • Chromatography
  • Electrophoresis
  • Spectroscopy

Protein Purification

• Highly purified protein is essential for determining its amino acid sequence and for use in biological, medical, and pharmaceutical processes
• Protein purification involves isolating a specific protein from a mixture of thousands of different proteins
• Classic approaches use differences in target protein properties, such as:
  • Salt precipitation (salting out)
  • Chromatographic techniques

Chromatographic Techniques

• Chromatographic separations partition molecules between two phases: mobile and stationary
• Stationary phase matrices interact with proteins based on their:
  • Charge
  • Hydrophobicity
  • Ligand-binding properties
• Types of chromatography include:
  • Ion exchange chromatography
  • Size exclusion chromatography
  • Affinity chromatography
  • High pressure chromatography (HPLC)

Ion Exchange Chromatography

• Proteins interact with the stationary phase by charge-charge interactions
• Proteins with a net positive charge adhere to beads with negatively charged functional groups (cation exchangers)
• Proteins with a net negative charge adhere to beads with positively charged functional groups (anion exchangers)

Affinity Chromatography

• Affinity chromatography exploits the high selectivity of proteins for their ligands
• Proteins can be purified using immobilized substrates, products, coenzymes, or inhibitors
• Bound proteins are eluted by competition with soluble ligand or by disrupting protein-ligand interactions using:
  • Urea
  • Guanidine hydrochloride
  • Mildly acidic pH
  • High salt concentrations

SDS-PAGE

• SDS-PAGE is a technique used to separate proteins based on their molecular weight
• It involves polyacrylamide gel electrophoresis in the presence of the anionic detergent sodium dodecyl sulfate (SDS)

Protein Analysis

• Protein structure can be analyzed using:
  • X-ray crystallography
  • Nuclear magnetic resonance (NMR)
  • Spectroscopy
• Protein activity and amount can be analyzed using:
  • UV spectroscopy
  • Bradford protein assay
  • Coomassie brilliant blue dye

Determination of Protein Primary Structure (Amino Acid Composition)

• Identification of proteins associated with specific physiologic states or diseases is an important goal of molecular medicine
• Primary sequence of a protein provides a molecular fingerprint for identification and information for cloning the gene or genes that encode it

Protein Sequencing

• N-Terminal analysis determines the amino terminal end of the polypeptide chain using:
  • Sanger's method
  • Edman's method
• C-Terminal analysis determines the carboxy terminal end of the polypeptide chain using:
  • Carboxypeptidase method
• Edman's sequencing method involves:
  • Chromatography
  • Electrophoresis
  • Spectroscopy

Protein Purification

• Highly purified protein is essential for determining its amino acid sequence and for use in biological, medical, and pharmaceutical processes
• Protein purification involves isolating a specific protein from a mixture of thousands of different proteins
• Classic approaches use differences in target protein properties, such as:
  • Salt precipitation (salting out)
  • Chromatographic techniques

Chromatographic Techniques

• Chromatographic separations partition molecules between two phases: mobile and stationary
• Stationary phase matrices interact with proteins based on their:
  • Charge
  • Hydrophobicity
  • Ligand-binding properties
• Types of chromatography include:
  • Ion exchange chromatography
  • Size exclusion chromatography
  • Affinity chromatography
  • High pressure chromatography (HPLC)

Ion Exchange Chromatography

• Proteins interact with the stationary phase by charge-charge interactions
• Proteins with a net positive charge adhere to beads with negatively charged functional groups (cation exchangers)
• Proteins with a net negative charge adhere to beads with positively charged functional groups (anion exchangers)

Affinity Chromatography

• Affinity chromatography exploits the high selectivity of proteins for their ligands
• Proteins can be purified using immobilized substrates, products, coenzymes, or inhibitors
• Bound proteins are eluted by competition with soluble ligand or by disrupting protein-ligand interactions using:
  • Urea
  • Guanidine hydrochloride
  • Mildly acidic pH
  • High salt concentrations

SDS-PAGE

• SDS-PAGE is a technique used to separate proteins based on their molecular weight
• It involves polyacrylamide gel electrophoresis in the presence of the anionic detergent sodium dodecyl sulfate (SDS)